This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of this project is to provide understanding of the roles of OST3/6 thiol oxidoreductases in Nlinked protein glycosylation and ER redox homeostasis. Thiol-dependent redox processes are involved in oxidative stress defense, signal transduction, and protein folding, modification and regulation, and are catalyzed by structurally distinct families of enzymes known as thiol oxidoreductases. Numerous ER thiol oxidoreductases involved in protein folding have been characterized;however, the overall machinery of folding and glycosylation remains poorly characterized. The N-linked protein glycosylation in the ER is an essential process and a key step in the control of protein folding in eukaryotes. OST3/6 proteins are abundant ER membrane-linked thioredoxin-fold thiol oxidoreductases involved in the redox control of N-linked protein glycosylation in the oligosaccharyltransferase complex. OST3/6 deficiency is associated with severe protein underglycosylation and ER stress. Homozygous deletion of human OST3/6 like protein, N33, correlates with metastatic prostate cancer and its allelic deletion is associated with human colorectal and pancreatic cancers. This observation suggests a possible tumor suppressor function of N33. In addition, there are two known cases of a natural knockout of N33 in humans which are associated with nonsyndromic mental retardation. In the proposed study, we will systematically characterize the biological function of OST3/6 proteins. The effect of OST3/6 deficiency will be examined with regard to efficiency of protein glycosylation and ER stress. Possible targets of OST3/6 proteins will be identified using thiol-mediated substrate-trapping method and global gene expression analysis. The OST3/6 roles in disulfide bond formation will be addressed in series of thiol oxidoreductase assays. These experiments will be carried in yeast Saccharomyces cerevisiae and mammalian cells. We also would like to develop OST3/6 knockout mouse models. These models will provide tools in a better understanding of the consequences of OST3/6 protein deficiency on cancer and brain disorders and will be useful in the analysis of OST3/6 biological function.